Hydrolytically stable, low- and high-pI isoelectric hydrogel membranes were prepared from poly(vinyl alcohol) (PVA) as alternatives to polyacrylamide-based isoelectric membranes that hydrolyze in acidic and basic solutions. Low-pI membranes were made by attaching an isoelectric buffer of a welldefined pI value (such as iminodiacetic acid, IDA, aspartic acid, ASP or glutamic acid, GLU) to the PVA backbone and crosslinking the PVA strands, in situ. The pH in these membranes does not change significantly with slight variations in the amount of isoelectric buffer that gets incorporated. The pI values of these membranes were pI is greater then 1.7 but less then 2.0 (IDAPVA), pI is greater then 2.0 but less then 2.6 (ASPPVA) and pI was greater then 2.6 but less then 3.4 (GLUPVA).The membranes were used as anodic membranes in isoelectric trapping (IET) experiments. Sugars, cyclodextrins (CDs), and certain polyhydroxy compounds have pKa values between 11.5 and 14. Thus, high-pI hydrogels were obtained by incorporating (i) quaternary ammonium derivatives of Beta-CD (QCDPVA) (ii) quaternary ammonium groups and Beta-CD (CDQPVA) and (iii) quaternary ammonium groups alone (QPVA) into the crosslinked PVA hydrogels. All three membranes had pI values greater than 11 and served as effective cathodic membranes for the IET of small ampholytic molecules and proteins. In pH-biased IET, proteins are collected into solutions of isoelectric buffers that set the pH to keep the proteins in a charged state affording high solubility and preventing precipitation. Thus, a series of isoelectric buffers (biasers) with high buffering capacity, high conductivity, and pI values covering the useful pH 2-10 range are needed. Two sets of such buffers were designed (i) with pI values between the pKa values of two carboxylic acid groups and (ii) with pI values between the pKa values of the conjugate acid form of two amine groups. Six of these buffers were synthesized and their synthesis was optimized. The products were obtained in their pure, isoelectric form and were extensively characterized.

No description available.

Isoelectric focusing is one of the most important techniques in protein separations. Preparative-scale isoelectric separations often use buffering membranes (isoelectric membranes), but there are no good known methods for the characterization of their pI values. Therefore, UV-absorbing carrier ampholyte mixtures (UVCAs) have been synthesized, analytically characterized, and utilized for the characterization of the pI value of a buffering membrane. To synthesize the UVCAs, addition of a UV-absorbing electrophile, 3-phenoxypropyl bromide (PhOPrBr), to a pentaethylenehexamine (PEHA) carrier ampholyte backbone, resulted in an intermediate that was subsequently reacted with increasing amounts of acrylic acid (up to 8 equiv) and itaconic acid (up to 2 equiv) via Michael’s addition. The intermediates and final products were characterized by 1H-NMR and full-column imaging capillary isoelectric focusing techniques. An optimal blended mixture of selected UVCAs was first desalted and purified by isoelectric trapping and its composition verified by full-column imaging isoelectric focusing. The mixture of UVCAs possessed a broad pI distribution from approximately pH 3 – 10. By isoelectric trapping, the mixture was separated into two subfractions with a polyacrylamide-based isoelectric membrane of known pI as the separation membrane and poly(vinyl) alcohol-based buffering membranes as the restriction membranes. The pI of the most basic UV-active carrier ampholyte in the anodic fraction was determined to be 4.4 and the pI of the most acidic UV-active carrier ampholyte in the cathodic fraction was determined to be 4.4, confirming that the pH of the polyacrylamide-based isoelectric membrane was pH 4.4.

Preparative-scale protein separations have always been critical to the advancement of the life sciences. Among preparative-scale separation techniques, isoelectric trapping (IET) promises efficient separations and high production rates. This dissertation focuses on the improvement of two aspects of preparative-scale IET protein separations: the instrumentation used and the monitoring of the separation. The first aspect (preparative-scale) is the IET device: the improvement of a multicompartmental electrolyzer (MCE) to increase the efficiency and production rate of IET separations. The redesign focused on three major areas: (1) the sealing system, (2) the configuration of the liquid flow path, and (3) the cooling system. The second aspect (analytical-scale) is the monitoring of the IET separation: the design and manufacture of durable surface-modified capillaries which provide controlled, variable anodic and cathodic electroosmotic flow (EOF) to help develop, plan, and monitor the IET separations.

Preparative-scale protein separations have always been critical to the advancement of the life sciences. Among preparative-scale separation techniques, isoelectric trapping (IET) promises efficient separations and high production rates. This dissertation focuses on the improvement of two aspects of preparative-scale IET protein separations: the instrumentation used and the monitoring of the separation. The first aspect (preparative-scale) is the IET device: the improvement of a multicompartmental electrolyzer (MCE) to increase the efficiency and production rate of IET separations. The redesign focused on three major areas: (1) the sealing system, (2) the configuration of the liquid flow path, and (3) the cooling system. The second aspect (analytical-scale) is the monitoring of the IET separation: the design and manufacture of durable surface-modified capillaries which provide controlled, variable anodic and cathodic electroosmotic flow (EOF) to help develop, plan, and monitor the IET separations.

The classical isoelectric trapping (IET) technique, using the multicompartment electrolyzer (MCE), has been one of the most successful electrophoretic techniques in preparative-scale protein separations. IET is capable of achieving high resolution discrimination of proteins, by isolating proteins in between buffering membranes, in their isoelectric state. However, due to the inherent nature of the IET process, IET has suffered several shortcomings which have limited its applicability. During a classical IET separation, a protein gets closer and closer to its pI value, thus the charge of the protein gets closer and closer to zero. This increases the likelihood of protein precipitation and decreases the electrophoretic velocity of the protein, thus making the separation very long. Furthermore, the problems are aggravated by the fact that the instrumentation currently used for IET is not designed to maximize the efficiency of electrophoretic separations. To address these problems, a new approach to IET has been developed, pH-biased IET. By controlling the solution pH throughout the separation, such that it is not the same as the protein’s pI values, the problems of reduced solubility and low electrophoretic migration velocity are alleviated. The pH control comes from a novel use of isoelectric buffers (also called auxiliary isoelectric agents or pH-biasers). The isoelectric buffers are added to the sample solution during IET and are chosen so that they maintain the pH at a value that is different from the pI value of the proteins of interest. Two new pieces of IET instrumentation have been developed, resulting in major improvements in protein separation rates and energy efficiency. A variety of separations, of both small molecules and proteins, have been successfully performed using the pH-biased IET principle together with the new instrumentation.

Isoelectric focusing is one of the most important techniques in protein separations. Preparative-scale isoelectric separations often use buffering membranes (isoelectric membranes), but there are no good known methods for the characterization of their pI values. Therefore, UV-absorbing carrier ampholyte mixtures (UVCAs) have been synthesized, analytically characterized, and utilized for the characterization of the pI value of a buffering membrane. To synthesize the UVCAs, addition of a UV-absorbing electrophile, 3-phenoxypropyl bromide (PhOPrBr), to a pentaethylenehexamine (PEHA) carrier ampholyte backbone, resulted in an intermediate that was subsequently reacted with increasing amounts of acrylic acid (up to 8 equiv) and itaconic acid (up to 2 equiv) via Michael’s addition. The intermediates and final products were characterized by 1H-NMR and full-column imaging capillary isoelectric focusing techniques. An optimal blended mixture of selected UVCAs was first desalted and purified by isoelectric trapping and its composition verified by full-column imaging isoelectric focusing. The mixture of UVCAs possessed a broad pI distribution from approximately pH 3 – 10. By isoelectric trapping, the mixture was separated into two subfractions with a polyacrylamide-based isoelectric membrane of known pI as the separation membrane and poly(vinyl) alcohol-based buffering membranes as the restriction membranes. The pI of the most basic UV-active carrier ampholyte in the anodic fraction was determined to be 4.4 and the pI of the most acidic UV-active carrier ampholyte in the cathodic fraction was determined to be 4.4, confirming that the pH of the polyacrylamide-based isoelectric membrane was pH 4.4.

Polyacrymalide gel isoelectric focusing (PAGIF) in thin-layer was used to resolve proteins of Mycoplasma spp., Acholeplasma spp. and of eight strains of Ureaplasma urealyticum (T-Strain). A mixture of urea, Triton X-100, and dithioerythritol was used to solubilize sonically disrupted cells.

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Isoelectric trapping (IET), has proven to be one of the most successful electrophoretic techniques used for separations of ampholytic compounds. IET is carried out in multicompartment electrolyzers (MCEs) in which adjacent compartments are joined through buffering membranes whose pH values bracket the pI of the ampholytic component to be trapped in the compartment. The present small-scale instruments use plastics as their structural materials, which causes poor Joule heat dissipation. The separation compartments have cylindrical or pear-shaped interiors with large internal diameters, which create long heat transfer paths. The long electrode distances yield low field strengths that lead to low electrophoretic velocities for the analytes. These factors interrelatedly limit the electric power that can be applied to the system, contributing to long separation times. Furthermore, these devices do not offer a realistic solution to the problems associated with the detection of low abundance proteins. To address these problems, two novel IET devices have been developed for small-scale IET separations. The first device, named MSWIFT, was constructed using thermally conductive, high-purity alumina as the structural material of the separation compartments. By creating narrow, 0.1- or 0.2-mL channels in thin alumina blocks, the heat transfer path from the center of the compartment to the wall was significantly decreased; and the distance between electrodes was greatly shortened. MSWIFT achieved 6 to 50 times faster IET separations compared to other MCEs. The second device, named ConFrac, was developed to simultaneously fractionate and concentrate ampholytic components from a complex sample into 0.1-mL collection compartments. By designing a system with a 2-dimensional pH gradient and allowing recirculation of the sample feed, the ConFrac demonstrated enrichment of analytes by a factor of 100 and greater.